Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples.

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.

Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells.

Here, we present DNA aptamers capable of specific binding to glial tumor cells in vitro, ex vivo, and in vivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, ex vivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show in vivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Nucleic Acid Aptamers Increase the Anticancer Efficiency and Reduce the Toxicity of Cisplatin-Arabinogalactan Conjugates In Vivo.

Cisplatin is an effective drug for treating various cancer types. However, it is highly toxic for both healthy and tumor cells. Therefore, there is a need to reduce its therapeutic dose and increase targeted bioavailability. One of the ways to achieve this could be the coating of cisplatin with polysaccharides and specific carriers for targeted delivery. Nucleic acid aptamers could be used as carriers for the specific delivery of medicine to cancer cells. Cisplatin-arabinogalactan-aptamer (Cis-AG-Ap) conjugate was synthesized based on Cis-dichlorodiammineplatinum, Siberian larch arabinogalactan, and aptamer AS-42 specific to heat-shock proteins (HSP) 71 kDa (Hspa8) and HSP 90-beta (Hsp90ab1). The antitumor effect was estimated using ascites and metastatic Ehrlich tumor models. Cis-AG-Ap toxicity was assessed by blood biochemistry on healthy mice. Here, we demonstrated enhanced anticancer activity of Cis-AG-Ap and its specific accumulation in tumor foci. It was shown that targeted delivery allowed a 15-fold reduction in the therapeutic dose of cisplatin and its toxicity. Cis-AG-Ap sufficiently suppressed the growth of Ehrlich's ascites carcinoma, the mass and extent of tumor metastasis in vivo. Arabinogalactan and the aptamers promoted cisplatin efficiency by enhancing its bioavailability. The described strategy could be very promising for targeted anticancer therapy.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

Development of Electrochemical Aptasensor for Lung Cancer Diagnostics in Human Blood.

We describe the preparation and characterization of an aptamer-based electrochemical sensor to lung cancer tumor markers in human blood. The highly reproducible aptamer sensing layer with a high density (up to 70% coverage) on the gold electrode was made. Electrochemical methods and confocal laser scanning microscopy were used to study the stability of the aptamer layer structure and binding ability. A new blocking agent, a thiolated oligonucleotide with an unrelated sequence, was applied to fill the aptamer layer's defects. Electrochemical aptasensor signal processing was enhanced using deep learning and computer simulation of the experimental data array. It was found that the combinations (coupled and tripled) of cyclic voltammogram features allowed for distinguishing between the samples from lung cancer patients and healthy candidates with a mean accuracy of 0.73. The capacitive component from the non-Faradic electrochemical impedance spectroscopy data indicated the tumor marker's presence in a sample. These findings allowed for the creation of highly informative aptasensors for early lung cancer diagnostics.

Proteomics-Based Regression Model for Assessing the Development of Chronic Lymphocytic Leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) is very ambiguous, showing either an indolent nature of the disease or having latent dangerous progression, which, if diagnosed, will require an urgent therapy. The prognosis of the course of the disease and the estimation of the time of therapy initiation are crucial for the selection of a successful treatment strategy. A reliable estimating index is needed to assign newly diagnosed CLL patients to the prognostic groups. In this work, we evaluated the comparative expressions of proteins in CLL blood cells using a label-free quantification by mass spectrometry and calculated the integrated proteomic indexes for a group of patients who received therapy after the blood sampling over different periods of time. Using a two-factor linear regression analysis based on these data, we propose a new pipeline for evaluating model development for estimation of the moment of therapy initiation for newly diagnosed CLL patients.

Proteomics-Based Machine Learning Approach as an Alternative to Conventional Biomarkers for Differential Diagnosis of Chronic Kidney Diseases.

Diabetic nephropathy, hypertension, and glomerulonephritis are the most common causes of chronic kidney diseases (CKD). Since CKD of various origins may not become apparent until kidney function is significantly impaired, a differential diagnosis and an appropriate treatment are needed at the very early stages. Conventional biomarkers may not have sufficient separation capabilities, while a full-proteomic approach may be used for these purposes. In the current study, several machine learning algorithms were examined for the differential diagnosis of CKD of three origins. The tested dataset was based on whole proteomic data obtained after the mass spectrometric analysis of plasma and urine samples of 34 CKD patients and the use of label-free quantification approach. The k-nearest-neighbors algorithm showed the possibility of separation of a healthy group from renal patients in general by proteomics data of plasma with high confidence (97.8%). This algorithm has also be proven to be the best of the three tested for distinguishing the groups of patients with diabetic nephropathy and glomerulonephritis according to proteomics data of plasma (96.3% of correct decisions). The group of hypertensive nephropathy could not be reliably separated according to plasma data, whereas analysis of entire proteomics data of urine did not allow differentiating the three diseases. Nevertheless, the group of hypertensive nephropathy was reliably separated from all other renal patients using the k-nearest-neighbors classifier "one against all" with 100% of accuracy by urine proteome data. The tested algorithms show good abilities to differentiate the various groups across proteomic data sets, which may help to avoid invasive intervention for the verification of the glomerulonephritis subtypes, as well as to differentiate hypertensive and diabetic nephropathy in the early stages based not on individual biomarkers, but on the whole proteomic composition of urine and blood.

Aptamer-Conjugated Superparamagnetic Ferroarabinogalactan Nanoparticles for Targeted Magnetodynamic Therapy of Cancer.

Nanotechnologies involving physical methods of tumor destruction using functional oligonucleotides are promising for targeted cancer therapy. Our study presents magnetodynamic therapy for selective elimination of tumor cells in vivo using DNA aptamer-functionalized magnetic nanoparticles exposed to a low frequency alternating magnetic field. We developed an enhanced targeting approach of cancer cells with aptamers and arabinogalactan. Aptamers to fibronectin (AS-14) and heat shock cognate 71 kDa protein (AS-42) facilitated the delivery of the nanoparticles to Ehrlich carcinoma cells, and arabinogalactan (AG) promoted internalization through asialoglycoprotein receptors. Specific delivery of the aptamer-modified FeAG nanoparticles to the tumor site was confirmed by magnetic resonance imaging (MRI). After the following treatment with a low frequency alternating magnetic field, AS-FeAG caused cancer cell death in vitro and tumor reduction in vivo. Histological analyses showed mechanical disruption of tumor tissues, total necrosis, cell lysis, and disruption of the extracellular matrix. The enhanced targeted magnetic theranostics with the aptamer conjugated superparamagnetic ferroarabinogalactans opens up a new venue for making biocompatible contrasting agents for MRI imaging and performing non-invasive anti-cancer therapies with a deep penetrated magnetic field.

Development of DNA Aptamers to Native EpCAM for Isolation of Lung Circulating Tumor Cells from Human Blood.

We selected DNA aptamers to the epithelial cell adhesion molecule (EpCAM) expressed on primary lung cancer cells isolated from the tumors of patients with non-small cell lung cancer using competitive displacement of aptamers from EpCAM by a corresponding antibody. The resulting aptamers clones showed good nanomolar affinity to EpCAM-positive lung cancer cells. Confocal microscopy imaging and spectral profiling of lung cancer tissues confirmed the same protein target for the aptamers and anti-EpCAM antibodies. Furthermore, the resulted aptamers were successfully applied for isolation and detection of circulating tumor cells in clinical samples of peripheral blood of lung cancer patients.

Aptamer-Targeted Plasmonic Photothermal Therapy of Cancer.

Novel nanoscale bioconjugates combining unique plasmonic photothermal properties of gold nanoparticles (AuNPs) with targeted delivery using cell-specific DNA aptamers have a tremendous potential for medical diagnostics and therapy of many cell-based diseases. In this study, we demonstrate the high anti-cancer activity of aptamer-conjugated, 37-nm spherical gold nanoparticles toward Ehrlich carcinoma in tumor-bearing mice after photothermal treatment. The synthetic anti-tumor aptamers bring the nanoparticles precisely to the desired cells and selectively eliminate cancer cells after the subsequent laser treatment. To prove tumor eradication, we used positron emission tomography (PET) utilizing radioactive glucose and computer tomography, followed by histological analysis of cancer tissue. Three injections of aptamer-conjugated AuNPs and 5 min of laser irradiations are enough to make the tumor undetectable by PET. Histological analysis proves PET results and shows lower damage of healthy tissue in addition to a higher treatment efficiency and selectivity of the gold nanoparticles functionalized with aptamers in comparison to control experiments using free unconjugated nanoparticles.

In Vivo Cancer Cells Elimination Guided by Aptamer-Functionalized Gold-Coated Magnetic Nanoparticles and Controlled with Low Frequency Alternating Magnetic Field.

Biomedical applications of magnetic nanoparticles under the influence of a magnetic field have been proved useful beyond expectations in cancer therapy. Magnetic nanoparticles are effective heat mediators, drug nanocarriers, and contrast agents; various strategies have been suggested to selectively target tumor cancer cells. Our study presents magnetodynamic nanotherapy using DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles exposed to a low frequency alternating magnetic field for selective elimination of tumor cells in vivo. The cell specific DNA aptamer AS-14 binds to the fibronectin protein in Ehrlich carcinoma hence helps deliver the gold-coated magnetic nanoparticles to the mouse tumor. Applying an alternating magnetic field of 50 Hz at the tumor site causes the nanoparticles to oscillate and pull the fibronectin proteins and integrins to the surface of the cell membrane. This results in apoptosis followed by necrosis of tumor cells without heating the tumor, adjacent healthy cells and tissues. The aptamer-guided nanoparticles and the low frequency alternating magnetic field demonstrates a unique non-invasive nanoscalpel technology for precise cancer surgery at the single cell level.

DNA Aptamers for the Characterization of Histological Structure of Lung Adenocarcinoma.

Nucleic acid aptamers are becoming popular as molecular probes for identification and imaging pathology and, at the same time, as a convenient platform for targeted therapy. Recent studies have shown that aptamers may be effectively used for tumor characterization and as commercially available monoclonal antibodies. Here we present three DNA aptamers binding to whole transformed lung cancer tissues, including tumor cells, connective tissues, and blood vessels. Protein targets have been revealed using affinity purification followed by mass spectrometry analyses, and they have been validated using a panel of correspondent antibodies and 3D imaging of tumor tissues. Each of the proteins targeted by the aptamers is involved in cancer progression and most of them are crucial for lung adenocarcinoma. We propose the use of these aptamers in aptahistochemistry for the characterization of the histological structure of lung adenocarcinoma. The value of the presented aptamers is their application together or separately for indicating the spread of neoplastic transformation, for complex differential diagnostics, and for targeted therapy of the tumor itself as well as all transformed structures of the adjacent tissues. Moreover, it has been demonstrated that these aptamers could be used for intraoperative tumor visualization and margin assessment.

Noninvasive Microsurgery Using Aptamer-Functionalized Magnetic Microdisks for Tumor Cell Eradication.

Magnetomechanical cell disruption using nano- and microsized structures is a promising biomedical technology used for noninvasive elimination of diseased cells. It applies alternating magnetic field (AMF) for ferromagnetic microdisks making them oscillate and causing cell membrane disruption with cell death followed by apoptosis. In this study, we functionalized the magnetic microdisks with cell-binding DNA aptamers and guided the microdisks to recognize cancerous cells in a mouse tumor in vivo. Only 10 min of the treatment with a 100 Hz AMF was enough to eliminate cancer cells from a malignant tumor. Our results demonstrate a good perspective of using aptamer-modified magnetic microdisks for noninvasive microsurgery for tumors.

Electrochemical aptasensor for lung cancer-related protein detection in crude blood plasma samples.

The development of an aptamer-based electrochemical sensor for lung cancer detection is presented in this work. A highly specific DNA-aptamer, LC-18, selected to postoperative lung cancer tissues was immobilized onto a gold microelectrode and electrochemical measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The aptamer protein targets were harvested from blood plasma of lung cancer patients by using streptavidin paramagnetic beads and square wave voltammetry of the samples was performed at various concentrations. In order to enhance the sensitivity of the aptasensor, silica-coated iron oxide magnetic beads grafted with hydrophobic C8 and C4 alkyl groups were used in a sandwich detection approach. Addition of hydrophobic beads increased the detection limit by 100 times. The detection limit of the LC-18 aptasensor was enhanced by the beads to 0.023 ng/mL. The formation of the aptamer - protein - bead sandwich on the electrode surface was visualized by electron microcopy. As a result, the electrochemical aptasensor was able to detect cancer-related targets in crude blood plasma of lung cancer patients.

Aptamers Selected to Postoperative Lung Adenocarcinoma Detect Circulating Tumor Cells in Human Blood.

Circulating tumor cells (CTCs) are rare cells and valuable clinical markers of prognosis of metastasis formation and prediction of patient survival. Most CTC analyses are based on the antibody-based detection of a few epithelial markers; therefore miss an important portion of mesenchymal cancer cells circulating in blood. In this work, we selected and identified DNA aptamers as specific affinity probes that bind to lung adenocarcinoma cells derived from postoperative tissues. The unique feature of our selection strategy is that aptamers are produced for lung cancer cell biomarkers in their native state and conformation without previous knowledge of the biomarkers. The aptamers did not bind to normal lung cells and lymphocytes, and had very low affinity to A549 lung adenocarcinoma culture. We applied these aptamers to detect CTCs, apoptotic bodies, and microemboli in clinical samples of peripheral blood of lung cancer and metastatic lung cancer patients. We identified aptamer-associated protein biomarkers for lung cancer such as vimentin, annexin A2, annexin A5, histone 2B, neutrophil defensin, and clusterin. Tumor-specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics.

DNA-aptamer targeting vimentin for tumor therapy in vivo.

In recent years, new prospects for the use of nucleic acids as anticancer drugs have been discovered. Aptamers for intracellular targets can regulate cellular functions and cause cell death or proliferation. However, intracellular aptamers have limited use for therapeutic applications due to their low bioavailability. In this work, we selected DNA aptamers to cell organelles and nucleus of cancer cells, and showed that an aptamer NAS-24 binds to vimentin and causes apoptosis of mouse ascites adenocarcinoma cells in vitro and in vivo. To deliver the aptamer NAS-24 inside cells, natural polysaccharide arabinogalactan was used as a carrier reagent. The mixture of arabinogalactan and NAS-24 was injected intraperitonealy for 5 days into mice with adenocarcinoma and inhibited adenocarcinoma growth more effectively than free arabinogalactan or the aptamer alone. The use of aptamers to intracellular targets together with arabinogalactan becomes a promising approach for anticancer therapy.

Development of bacteriostatic DNA aptamers for salmonella.

Salmonella is one of the most dangerous and common food-borne pathogens. The overuse of antibiotics for disease prevention has led to the development of multidrug resistant Salmonella. Now, more than ever, there is a need for new antimicrobial drugs to combat these resistant bacteria. Aptamers have grown in popularity since their discovery, and their properties make them attractive candidates for therapeutic use. In this work, we describe the selection of highly specific DNA aptamers to S. enteritidis and S. typhimurium. To evolve species-specific aptamers, twelve rounds of selection to live S. enteritidis and S. typhimurium were performed, alternating with a negative selection against a mixture of related pathogens. Studies have shown that synthetic pools combined from individual aptamers have the capacity to inhibit growth of S. enteritidis and S. typhimurium in bacterial cultures; this was the result of a decrease in their membrane potential.